Summary My lab's work has been at the forefront of studies showing that nuclear organization and long-range chromatin interactions play an essential role in recombination and gene regulation. In this application we have incorporated two distinct projects that extend this work. The first project focuses on understanding the mechanisms underlying feedback control of RAG activity in individual lymphocytes and the consequence of de-regulated cleavage. V(D)J recombination has to be tightly regulated to ensure that cleavage does not continue in cis, or in trans on accessible target loci that undergo recombination at overlapping stages of development as well as on actively transcribed off-target loci with cryptic recombination signal sequence (RSS) sites that bind the RAG proteins. Our recent studies reveal that ATM and the C terminus of RAG2 have an important role in feedback control of cleavage in individual cells through modulation of nuclear organization. This limits the number of potential substrates for translocation and provides an important mechanism for protecting genome stability. Given that an absence of the C terminus of RAG2 and inhibition of ATM kinase activity lead to similar phenotypes we hypothesized they could act in the same pathway. In our most recent unpublished work we identified a conserved SQ target phosphorylation site on RAG2 (residues 365-366) that recapitulates the function of the RAG2 C-terminus and ATM in regulating cleavage. However, in contrast to these two mutants, RAG2-S356A has a stable RAG post cleavage complex. Thus, for the first time we have a tool to study feedback regulation in the absence of any confounding repair defect. Here we aim to determine!(i) the mechanism underlying feedback control of RAG activity in individual cells, (ii) the consequences of cleavage deregulation on allelic exclusion, genome instability and gene regulation and (ii) the mechanism by which deregulated RAG activity contributes to oncogenesis. The second project focuses on understanding the mode of action of enhancers in controlling gene regulation in the context of 3D chromatin structure. Enhancers play a fundamental role in ensuring precise control of transcriptional patterns in development and differentiation. Physical contacts between genes and these regulatory elements are essential for proper transcriptional control and maintenance of these interactions is critical for preventing aberrations in physiological processes that could manifest as disease states. Using new tools developed under the support of GM086852 and GM112192 we are now able to investigate these interactions using live imaging systems and high-resolution chromosome conformation capture (4C). Specifically, our aim is to investigate the mode of action of enhancers in the context of: (i) enhancers that control the regulation of more than one target gene, (ii) the functional relevance of clusters of enhancers that constitute super-enhancers in regulating target loci and the factors that contribute to their evolution, and finally (iii) the potential role of transposable elements in driving gene regulatory networks in health and disease.